The present invention relates to methods for identifying novel compounds for use in the treatment of cancer and to compounds inhibiting the MyD88/Erk MAP kinase interaction for treating cancer.
It is well established that inflammation acts as a promoter of carcinogenesis. MyD88, an adaptor protein in the TLR signaling pathway, has therefore been implicated in carcinogenesis through its role in inflammation.
The present invention now surprisingly shows that MyD88 plays an intrinsic role in ras-dependent carcinogenesis in vitro and in vivo. Specifically, MyD88 binds ERK MAP Kinase through a conserved motif, blocking therefore the inactivation of ERK by its specific phosphatase MKP3, and leading to the amplification of the activity of the ras canonical pathway. The relevance of this mechanism in human cancers is demonstrated, MyD88 appears overexpressed and associated to phospho-ERK in many human cancers. Moreover, the experimental data shows that MyD88 participates directly in the ras signaling pathway and in the malignant transformation induced by this oncogene.
Inhibitors of the MyD88/Erk MAP Kinase interaction induce apoptosis in tumor cells. Interestingly, a synergistic effect is observed between inhibitors of the MyD88/Erk MAP kinase and DNA damage inducing chemotherapy. The present invention therefore provides new therapeutic perspectives in cancers, where the first line therapy is based on DNA damage inducing chemotherapy agents. Combination of DNA damage inducing chemotherapy agents with inhibition of the MyD88/Erk MAP kinase interaction enables dose reduction in sensitive tumors and therefore increases patient tolerance or re-induces sensitivity in resistant cancer cell lines having highly efficient DNA repair mechanisms.
1) The present invention is related to methods for selecting in vitro compounds capable of potentiating the effect of a DNA damage inducing chemotherapy agent for the treatment of cancer comprising the following steps:
a) Providing at least one candidate compound;
b) Contacting at least one candidate compound with a ERK MAPK protein and a MyD88 protein, under conditions suitable to allow ERK MAPK and MyD88 to interact in the absence of the candidate molecule;
c) Determining the interaction of ERK MAPK and MyD88 as measured in the presence and in the absence of at least one candidate compound; and
d) Selecting a candidate compound inhibiting the interaction of ERK MAPK and MyD88.
2) Method for selecting in vitro compounds capable of potentiating the effect of a DNA damage inducing chemotherapy agent for the treatment of cancer according to 1, wherein one or both of ERK MAPK and MyD88 are attached to a detectable label.
3) Method for selecting in vitro compounds capable of potentiating the effect of a DNA damage inducing chemotherapy agent for the treatment of cancer according to anyone of 1-2, wherein the interaction of ERK MAPK and MyD88 is determined using a two-hybrid system, affinity chromatography, co-immunoprecipitation, subcellular fractionation and isolation of large molecular complexes, immunoblotting, immunolabelling, a proximity ligation assay, an immunoprecipitation assay, a biacore assay or a GST pull-down assay.
4) Method for selecting in vitro compounds capable of potentiating the effect of a DNA damage inducing chemotherapy agent for the treatment of cancer according to anyone of 1-3, wherein the DNA damage inducing chemotherapy agent is selected in the group consisting of oxaliplatin, cisplatin, carboplatin, doxorubicin, etoposide, bleomycin, and mixtures thereof.
5) Method for selecting in vitro compounds capable of potentiating the effect of a DNA damage inducing chemotherapy agent for the treatment of cancer according to anyone of 1-4, wherein the cancer is selected in the group consisting of colorectal cancer, intestinal cancer, melanoma, lung cancer and cervical cancer.
6) Method for selecting in vitro compounds capable of potentiating the effect of a DNA damage inducing chemotherapy agent for the treatment of cancer comprising the following steps:
a) Providing at least one candidate compound;
b) Providing transformed cells expressing stably MyD88 under the control of an inducible promoter and expressing a reporter gene under the control of a promoter from the MAPK pathway selected from the SRE promoter, the ELK promoter and the MyC promoter;
c) Inducing overexpression of MYD88 in said transformed cells in the presence of at least one candidate compound;
d) Measuring the expression of the reporter gene and selecting at least a candidate compound inhibiting the expression of the reporter gene;
e) Providing immortalized fibroblasts transformed by transfection with the MyD88 and Myc oncogenes;
f) Monitoring focus formation in the presence of at least one candidate compound selected in step d) and in the presence of at least one DNA damage inducing chemotherapy agent, and selecting a candidate compound potentiating the inhibitory effect of the DNA damage inducing chemotherapy agent on focus formation.
7) Method for selecting in vitro compounds capable of potentiating the effect of a DNA damage inducing chemotherapy agent for the treatment of cancer according to 6, wherein in step b) the promoter from the MAPK pathway is the SRE promoter.
8) Method for selecting in vitro compounds capable of potentiating the effect of a DNA damage inducing chemotherapy agent for the treatment of cancer according to anyone of 6-7, wherein the method further comprises the following steps:                Providing transformed cells expressing stably MyD88 under the control of an inducible promoter and expressing a reporter gene under the control of a promoter from the inflammation pathway selected from the Nf-kB promoter and the ISRE promoter;        Inducing overexpression of MYD88 in said transformed cells in the presence of at least one candidate compound;        Measuring the expression of the reporter gene and selecting a candidate compound which does not inhibit the expression of the reporter gene.        
9) Method for selecting in vitro compounds capable of potentiating the effect of a DNA damage inducing chemotherapy agent for the treatment of cancer according to 8, wherein the promoter from the inflammation pathway is Nf-kB.
10) Method for selecting in vitro compounds capable of potentiating the effect of a DNA damage inducing chemotherapy agent for the treatment of cancer according to anyone of 6-9, wherein in step b) the cells are selected from the HCT116 cell line, the A375 cell line and a HeLa cell line.
11) Method for selecting in vitro compounds capable of potentiating the effect of a DNA damage inducing chemotherapy agent for the treatment of cancer according to anyone of 6-10, wherein the inducible promoter is an antibiotic-inducible promoter.
12) Method for selecting in vitro compounds capable of potentiating the effect of a DNA damage inducing chemotherapy agent for the treatment of cancer according to anyone of 6-11, wherein the reporter gene is luciferase.
13) Method for selecting in vitro compounds capable of potentiating the effect of a DNA damage inducing chemotherapy agent for the treatment of cancer according to anyone of 6-12, wherein the immortalized fibroblasts are NIH3T3 immortalized fibroblasts.
14) Method for selecting in vitro compounds capable of potentiating the effect of a DNA damage inducing chemotherapy agent for the treatment of cancer according to anyone of 6-13, wherein the cancer is selected in the group consisting of colorectal cancer, intestinal cancer, melanoma, lung cancer and cervical cancer.
15) Method for selecting in vitro compounds capable of potentiating the effect of a DNA damage inducing chemotherapy agent for the treatment of cancer according to anyone of 6-14, wherein the DNA damage inducing chemotherapy agent is selected from oxaliplatin, cisplatin, carboplatin, doxorubicin, etoposide and mixtures thereof.
16) Method for selecting in vitro compounds capable of potentiating the effect of a DNA damage inducing chemotherapy agent for the treatment of cancer according to anyone of 6-14, wherein the at least one candidate compound of step a) is selected according to a method according to anyone of 1-5.
The present invention is related to an in vitro method for selecting a compound for the treatment of cancer comprising the following steps:
a) Providing candidate compounds;
b) Providing transformed cells expressing stably MyD88 under the control of an inducible promoter and expressing a reporter gene under the control of a promoter from the MAPK pathway selected from the SRE promoter, the ELK promoter and the MyC promoter;
c) Inducing overexpression of MYD88 in said transformed cells in the presence of the candidate compound;
d) Measuring the expression of the reporter gene and selecting a candidate compound inhibiting the expression of the reporter gene;
e) Providing immortalized fibroblasts transformed by transfection with the MyD88 and Myc oncogenes;
f) Monitoring focus formation in the presence of the candidate compound selected in step d) and selecting said compound if focus formation is inhibited. Preferably, in the methods of the present invention the promoter from the MAPK pathway in step b) is the SRE promoter.
In preferred embodiments, the methods of the present invention further comprise the following steps:                Providing transformed cells expressing stably MyD88 under the control of an inducible promoter and expressing a reporter gene under the control of a promoter from the inflammation pathway selected from the Nf-kB promoter and the ISRE promoter;        Inducing overexpression of MYD88 in said transformed cells in the presence of the candidate compound;        Measuring the expression of the reporter gene and selecting a candidate compound which does not inhibit the expression of the reporter gene.Preferably, the promoter from the inflammation pathway is the Nf-kB promoter.        
In preferred embodiments, the cells are selected from the HCT116 cell line, the A375 cell line and a HeLa cell line in the methods of the present invention. Preferably, the inducible promoter is an antibiotic-inducible promoter. Preferably, the reporter gene is luciferase. In the methods of the present invention, the immortalized fibroblasts are preferably NIH3T3 immortalized fibroblasts.
The present invention is also related to an in vitro method for selecting a compound for the treatment of cancer comprising determining whether said compound inhibits the interaction between the MyD88 protein and the ERK MAP kinase by a proximity ligation assay, an immunoprecipitation assay, a biacore assay, a GST pull-down assay or a yeast two hybrid system. The present invention is also related to a compound inhibiting the interaction of MyD88 and ERK MAP kinase selected from peptides inhibiting the interaction of MyD88 with ERK MAP kinase and interfering RNA inhibiting the expression of human Myd88, for use in the treatment of cancer by inducing apoptosis in cancer cells. Preferably, the compounds according to the present invention are for use in treatment of cancers selected from colorectal cancer, intestinal cancer, melanoma, lung cancer and cervical cancer. Advantageously, the cancer is colorectal cancer.
The present invention also relates to a composition comprising a DNA damage inducing chemotherapy agent and a compound inhibiting the interaction of MyD88 with ERK MAP kinase selected from peptides inhibiting the interaction of MyD88 with ERK MAP kinase and interfering RNA inhibiting the expression of human Myd88, for use in the treatment of cancer. The present invention further relates to a composition comprising a compound inhibiting the interaction of MyD88 with ERK MAP kinase selected from peptides inhibiting the interaction of MyD88 with ERK MAP kinase and interfering RNA inhibiting the expression of human Myd88, for use in combination with DNA damage inducing chemotherapy agents in the treatment of cancer. In the methods and compositions of the present invention, the DNA damage inducing chemotherapy agent is preferably selected from oxaliplatin, cisplatin, carboplatin, doxorubicin, etoposide and mixtures thereof. The present invention is also directed to kits of part comprising a DNA damage inducing chemotherapy agent and a compound inhibiting the interaction of MyD88 with ERK MAP kinase selected from peptides inhibiting the interaction of MyD88 with ERK MAP kinase and interfering RNA inhibiting the expression of human Myd88, for use in the treatment of cancer.